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Establishment and characterization of mouse tumor cell line with irreversible downregulation of MHC class I molecules
Lhotáková, Karolína ; Poláková, Ingrid (advisor) ; Černý, Jan (referee)
Malignant diseases are one of the major causes of death worldwide and the most common reason is the ability of tumor cells to escape the immune system. One of the main mechanisms used by tumors to avoid the immune response is downregulation of MHCI expression. In this thesis, the preparation of tumor cell line with irreversible downregulation of B2m is described. Inactivation of B2m was confirmed by flow cytometry. However, MHCI heavy chain H2-Db were expressed at the cell surface in a small amount after stimulation with IFNγ. In vitro proliferation of this clone was decreased. The tumor formation was delayed, and this effect was mediated by NK cells that played the major role in antitumor immunity. Metastatic activity of these cells was not affected after inactivation of B2m. TC-1/dB2m cells are not sensitive to DNA immunization. In combined immunotherapy experiments with DNA vaccinaton and ODN1826 adjuvant, a weak deceleration of tumor growth was achieved repeatedly. TC-1/dB2m induced tumors contained more CD4+ T lymphocytes, Treg, γδ T lymfocytes and plasmacytoid dendritic cells, and fewer NK cells and macrophages than tumors induced by original TC-1 cells. Moreover, combined immunotherapy did not increase the infiltration of the TC-1/dB2m tumors with immune cells. The TC-1/dB2m tumor cell line...
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Functions of open MHC class I molecule conformations
Vaníčková, Marie ; Poláková, Ingrid (advisor) ; Vaníková, Šárka (referee)
The major role of MHC class I molecules in adaptive system is to present antigen peptides derived from intracellular environment on the cell surface. These peptides are recognized by CD8+ T-lymphocytes and they can also interact with NK cells via trans-interaction. MHC class I molecules are composed of a heavy chain, β2-microglobulin (β2m, light chain) and peptide, forming a closed conformation. The heavy chain is non-covalently associated with the light chain and is folded into extracellular domain (α1, α2, α3 subunits), transmembrane domain and cytoplasmic domain (with conserved motifs). Upon active metabolism, the β2m and peptide may dissociate from the MHC I heavy chain what leads to the formation of open conformations of MHC I. This conformational change causes the subunit to unfold and allow its interaction with various receptors and molecules. Open conformers of MHC I may form cis-interactions with themselves creating homodimers involved in immunological functions or they can associate with different receptors on the cell surface creating heterodimers responsible for non-immunological functions. Soluble forms of free heavy chains also exist outside of the cell surface. Cis-associations are very important as they influence signaling pathways of the cell, inhibition or activation of...
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The role of IFI16 protein in viral infection
Karchňák, Jan ; Šroller, Vojtěch (advisor) ; Poláková, Ingrid (referee)
6 Abstract Viruses are obligate intracellular parasites causing a large variety of diseases. Most of their hosts, including humans, have developed particular mechanisms, which are meant to tackle such diseases. First line of defense against viruses are pattern recognition receptors. These receptors are responsible for detection of pathogen associated molecular patterns (PAMPs) and of damage associated molecular patterns (DAMPs). Inteferon γ inducible protein 16 (IFI16) is one of these receptors and is responsible for detecting alien and damaged DNA both in the nucleus and cytoplasm. Its structure contains two sequence independent DNA binding HIN domains and one PYD domain, which mediates its protein-protein interactions. In the cell it functions as a regulator of cell cycle, differentiation and plays a role in cell aging. IFI16 also triggers activation of non-specific immune response and it directly acts as a restrictive factor for many viruses. During evolution these viruses have evolved mechanism which they us to evade its imunne activity or even use it to their advantage. Keywords: IFI16, STING, DNA sensing, interferons, restriction factor, pattern recogniton receptors
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The role of cellular sensors in viral infection
Karchňák, Jan ; Šroller, Vojtěch (advisor) ; Poláková, Ingrid (referee)
Viruses are obligate intracellular parasites causing a large variety of diseases. Most of their hosts, including humans, have developed particular mechanisms, which are meant to tackle such diseases. First line of defense against viruses are germline encoded Pattern recognition receptors. These receptors are responsible for detection of pathogen associated molecular patterns (PAMPs) and of damage associated molecular patterns (DAMPs). Inteferon γ inducible protein 16 (IFI16) is one of these receptors and is responsible for detecting alien and damaged DNA both in the nucleus and cytoplasm. Its structure contains two sequence independent DNA binding HIN domains and one PYD domain, which mediates its protein-protein interactions. In the cell it functions as a regulator of cell cycle and differentiation. It also has telomerase activity and regulates cell senescence. IFI16 also triggers activation of non-specific immune response and it directly acts as a restrictive factor for many viruses. During evolution these viruses have evolved mechanism which they us to evade its imunne activity or even use it to their advantage. Keywords: IFI16, STING, DNA sensing, interferons, restriction factor, pattern recogniton receptors
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Gene Immunotherapy of Cancer: DNA Vaccines against HPV 16
Poláková, Ingrid
of PhD. Thesis Gene Immunotherapy of Cancer: DNA Vaccines against HPV 16 Mgr. Ingrid Poláková Cervical carcinoma (CC) represents the second most frequent cancer in women, mostly associated with human papillomavirus (HPV) infection. Nowadays, two prophylactic vaccines, protecting against HPV 16 and HPV 18, are licensed. Nevertheless, development of therapeutic vaccines is desirable to eliminate current HPV infections and to treat progressing tumours. Suitable targets for vaccination are viral E6 and E7 oncoproteins. Since its discovery, DNA vaccination has become an effective strategy for development of vaccines against cancer including CC. Unfortunately, the immunogenicity of DNA vaccines in large animals and particularly in humans is low. Therefore, several ongoing studies are focused on strategies enhancing the efficacy and safety of DNA vaccines. In this work, the immunogenicity of DNA vaccines against HPV 16 delivered by a gene gun was evaluated after the fusion of the E7 and E6 genes with GUS. The increased steady-state level of the E7GGG.GUS deletion mutants and the GUS.E7GGG fusion protein enhanced the production of E7-specific antibodies after immunisation with these vaccines but did not improve the CTL response. Joining of the signal sequence with GUS.E7GGG led to ER-localisation of the...
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Establishment and characterization of mouse tumor cell line with irreversible downregulation of MHC class I molecules
Lhotáková, Karolína ; Poláková, Ingrid (advisor) ; Černý, Jan (referee)
Malignant diseases are one of the major causes of death worldwide and the most common reason is the ability of tumor cells to escape the immune system. One of the main mechanisms used by tumors to avoid the immune response is downregulation of MHCI expression. In this thesis, the preparation of tumor cell line with irreversible downregulation of B2m is described. Inactivation of B2m was confirmed by flow cytometry. However, MHCI heavy chain H2-Db were expressed at the cell surface in a small amount after stimulation with IFNγ. In vitro proliferation of this clone was decreased. The tumor formation was delayed, and this effect was mediated by NK cells that played the major role in antitumor immunity. Metastatic activity of these cells was not affected after inactivation of B2m. TC-1/dB2m cells are not sensitive to DNA immunization. In combined immunotherapy experiments with DNA vaccinaton and ODN1826 adjuvant, a weak deceleration of tumor growth was achieved repeatedly. TC-1/dB2m induced tumors contained more CD4+ T lymphocytes, Treg, γδ T lymfocytes and plasmacytoid dendritic cells, and fewer NK cells and macrophages than tumors induced by original TC-1 cells. Moreover, combined immunotherapy did not increase the infiltration of the TC-1/dB2m tumors with immune cells. The TC-1/dB2m tumor cell line...
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Indoleamine 2,3-dioxygenase 1 inhibitors in cancer immunotherapy
Muffová, Barbora ; Poláková, Ingrid (advisor) ; Šroller, Vojtěch (referee)
The indoleamine 2,3-dioxygenase (IDO 1) enzyme is expressed in small amounts in most of the mammalian tissues, and its production is detected also in various types of tumours. IDO 1 catalyses the very first step of the kynurenine pathway, the tryptophan conversion to N-formylkynurenine, which is further metabolized to kynurenine (Kyn). The kynurenine/ tryptophan ratio (kyn/trp) may be used as a prognostic marker in research and treatment of IDO 1+ tumours. The kyn/trp demonstrates the activity of IDO 1 in tumours. The goal of cancer immunotherapy based on IDO 1 inhibition is to reverse or reduce the protumour effects of IDO 1, such as avoiding NK and T cells inhibition and activation of regulatory T cells or association with tumour-associated macrophages (TAM). IDO 1 inhibitors have been examined alone in monotherapy or together with cytotoxic T-lymphocytes antigen 4 (CTLA-4) inhibitors and programmed cell death protein 1 (PD-1) inhibitors in combined therapy. Recently, several studies are dedicated to invent inhibitors, which are able to inhibit the activity of other trp-catalysing enzymes, the indoleamine 2,3-dioxygenase 2 (IDO 2) and tryptophan 2,3-dioxygenase (TDO), together with the IDO 1 activity. Cancer immunotherapy based on IDO 1 inhibition may be combined also with chemotherapy.
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Dengue vaccines
Holšteinová, Aneta ; Šroller, Vojtěch (advisor) ; Poláková, Ingrid (referee)
Since the middle of the 20th century the number of people infected by the dengue virus (DENV) has been constantly increasing. Specific antiviral drugs are not available, so the objective for eliminating transmission has become the construction of an effective and safe vaccine with an emphasis on inducing a balanced immune reaction against all of the DENV serotypes. Momenteraly, the only licenced vaccine is CYD-TDV (Chimeric Yellow Fever- Dengue, Live-Attenuated, Tetravalent Dengue Vaccine). This vaccine uses the structure of the effective and safe vaccine against the yellow fever virus (YFV) from which surface protein sequences are substituted for corresponding DENV sequences. While preclinical studies displayed promising results, complications have arised during clinical studies, thus creating limiting criteria for their use. For this reason the search for new vaccines that could ensure better safety from DENV for a wider range of people and replace CYD-TDV is ongoing. In the III. phase of the clinical studies there are two live-attenuated vaccines (TetraVax-DV and DENVax). The subunite vaccine (V180), DNA vaccine (TVDV) or inactivated vaccine (TDENV PIV) was forwarded into the I. phase. The combination of several vaccine schedules is also being used (tetravalent live-attenuated...
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Modification of murine tumor cell lines with CRISPR/Cas9 system and their characterization
Lhotáková, Karolína ; Poláková, Ingrid (advisor) ; Brábek, Jan (referee)
MHCI molecules are constitutively expressed in all nucleated cells and play a key role in antigen presentation to CD8+ T lymphocytes. One of the tumor immune evasion strategies is MHCI expression downregulation. This leads to an impaired recognition of tumor antigens by CD8+ T lymphocytes that are unable to start the immune response. Since the MHCI expression downregulation occurs in up to 90 % of some tumors it is neccesary to have a clinical relevant tumor model without a MHCI surface expression that would be used for testing of immunotherapeutic approaches. This thesis describes a production of new model cell lines of TC-1 tumor cells with irreversibly downregulated MHCI. That was achieved by an inactivation of B2m, which is a part of MHCI, by gene editing using CRISR/Cas9. The B2m inactivation was confirmed by flow cytometry, western blot and sanger sequencing of single alleles. The inactivation slowed down the cell growth for both in vitro and in vivo. The cell metastatic activity was not affected. The tumors established by cells without the B2m expression are not sensitive to DNA vaccine against HPV16 E7 oncoprotein by a pBSC/PADRE.E7GGG vaccine. The main effector function against these tumors possess the NK1.1+ cells. In a therapeutic vaccination experiment it was repeatedly achieved of...
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Role of molecular chaperones Hsp70 and Hsp90 in the replication cycle of DNA viruses
Žáčková, Sandra ; Horníková, Lenka (advisor) ; Poláková, Ingrid (referee)
Molecular chaperones are proteins which enable other proteins to assemble into native conformation and are essential for viability of the cells. Chaperones of the Hsp70 family bind to newly synthetized and denaturated proteins, prevent their aggregation and facilitate their assembly. They participate in assembly and disassembly of oligomers and also in the transport across the membranes. Chaperones of the Hsp90 family do not participate in the assembly of nascent or denaturated proteins. They bind proteins which are nearly in native conformation and enable them to assemble into conformation suitable for ligand binding or interacting with other proteins. These attributes predestinate chaperones to participate in the replication cycle of DNA viruses. A huge amount of proteins is translated during viral infection. These proteins require the chaperones to facilitate their assembly and are also required for assembly into oligomers and macromolecular structures. In addition to capsid assembly the chaperones also participate in transport of genetic information to the sites of replication, disassembly of incoming viral particles or replication of viral DNA. Therefore, the development of specific chaperone inhibitors is a promising approach. They could be used against broad spectrum of viral infections...
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